Benzylamines as highly potent inhibitors of the sterol biosynthesis pathway in Leishmania amazonensis leading to oxidative stress and ultrastructural alterations

Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus. Benzylamines are a class of compounds selectively designed to inhibit the squalene synthase (SQS) that catalyzes the first committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzylamines (SBC 37–43) against Leishmania amazonensis. After the first screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating high selectivity. Analysis of the sterol composition revealed a depletion of endogenous 24-alkylated sterols such as episterol and 5-dehydroepisterol, with a concomitant accumulation of fecosterol, implying a disturbance in cellular lipid content. This result suggests a blockade of de novo sterol synthesis at the level of SQS and C-5 desaturase. Furthermore, physiological analysis and electron microscopy revealed three main alterations: (1) in the mitochondrion; (2) the presence of lipid bodies and autophagosomes; and (3) the appearance of projections in the plasma membrane. In conclusion, our results support the notion that benzylamines have a potent effect against Leishmania amazonensis and should be an exciting novel pharmaceutical lead for developing new chemotherapeutic alternatives to treat leishmaniasis.

Cell viability and cytotoxicity assays. For primary screening of the antileishmanial effects of the SBCs 37-43, we evaluated the cell viability and cytotoxicity effects in L. amazonensis promastigotes and peritoneal macrophages using the CellTiter 96 ® Aqueous MTS Assay (Promega, United States) 16,29 . For analysis in promastigotes, we started the culture at a cell density of 1 × 10 6 cells/ml in Warren's medium supplemented with 10% FBS. After 24 h, different concentrations of  were added to the cultures. Cell viability and the cytotoxic effects were measured at 24, 48, and 72 h of treatment, when all groups, including untreated, were transferred to a transparent 96-well plate in triplicate. MTS/PMS assay reaction was quantified by optical density measurement at 490 nm in a microplate reader and SpectraMax M 2 /M 2 e spectrofluorometer (Molecular Devices, United States). As a negative control, parasites were fixed with 0.4% nascent formaldehyde for 10 min at room temperature before the incubation. Cytotoxicity effects of SBCs 37-43 in murine macrophages were also evaluated using the same MTS/PMS assay reaction described above. After washing the peritoneal cavity of the BALB/c mice with Hanks's solution, murine macrophages were obtained and cultivated in a transparent 96-well plate with RPMI medium supplemented with 10% FBS and maintained at 37 °C in 5% CO 2 . After 24 h of cultivation, SBCs 37-43 were added at different concentrations. Macrophage viability was measured at 24, 48, and 72 h of treatment. MTS/PMS assay reaction was also quantified by optical density measurement at 490 nm in a microplate reader and SpectraMax M 2 /M 2 e spectrofluorometer (Molecular Devices, United States). The cytotoxicity concentration to reduce 50% of viable macrophages (CC 50 ) was determined. www.nature.com/scientificreports/ Growth inhibition of promastigotes and amastigotes of L. amazonensis. After evaluating the cell viability and cytotoxic effects by MTS assay in promastigote forms, we also analyzed the effects of the SBCs 37-43 in the growth of promastigotes. For this, promastigote cultures were initiated at a cell density of 1.0 × 10 6 cells/ml. After 24 h of growth, SBCs 37-43 were added at different concentrations from concentrated stock solutions, and cell densities were evaluated daily over 96 h of growth using a Neubauer chamber. Based on the analysis of CC 50 and IC 50 in promastigotes, and the cytotoxic effects in murine macrophages, three benzyl farnesyl amine mimetics (SBCs 37, 39, and 40) were chosen for evaluation against intracellular amastigotes. To evaluate the effects of compounds on L. amazonensis intracellular amastigotes, macrophages were infected as described previously and incubated with different concentrations of compounds after 24 h of infection. Fresh medium was added daily until 3 days (24,48, and 72 h of treatment). After this time, cultures were fixed in Bouin's solution 17 , washed with 70% ethanol, distilled water, and then stained with Giemsa solution for 1 h. The number of intracellular amastigotes was obtained after the count in light microscopy. Association indexes ((mean number of parasites internalized X percentage of infected macrophages)/total number of macrophages) were determined and used as a parameter to calculate the percentage of infection for each condition used in this study. The concentration that inhibited 50% of growth (IC 50 ) and selective index (SI) was calculated. The CC 50 and IC 50 were calculated by fitting the values to a non-linear curve analysis. The regression analyses were performed with SigmaPlot 10 software.
Estimation of the mitochondrial transmembrane electric potential. Mitochondrial transmembrane electric potential (Δψ m ) of the untreated and treated promastigotes was analyzed using the JC-1 fluorochrome (Molecular Probes, United States). This lipophilic and cationic mitochondrial vital dye accumulates in  www.nature.com/scientificreports/ the mitochondria in response to Δψ m since its fluorescence indicates an energized mitochondrial state 15 . JC-1 exists as a J-monomer that, in the absence of Δψ m , accumulates in low concentration with emission wavelength at 530 nm (green fluorescence); however, in the presence of Δψ m , JC-1 accumulates as J-aggregates with emission at 590 nm (red fluorescence). Parasites were prepared as previously described 15,16 . For each sample, 1 × 10 7 parasites were incubated with 10 μg/ml JC-1 for 25 min, with readings made every minute using a microplate reader and spectrofluorometer SpectraMax M2/M2 e (Molecular Devices, United States). As a positive control for the mitochondrial membrane depolarization, cells were incubated with 2 μM FCCP (a mitochondrial protonophore) during the initial 20 min of reading. After 20 min, 2 μM FCCP was added to all samples to abolish the Δψ m . The relative Δψ m values were obtained, calculating the ratio between the reading at 590 nm and 530 nm (590:530 nm). Experiments were independently repeated at least three times in triplicate, and the graphic shows the mean and standard deviation of one representative experiment.
Evaluation of ROS production. As described previously, intracellular ROS levels were evaluated in control and compound-treated promastigotes 17  Electron tomography. For electron tomography, ribbons of 200 nm thick serial sections were obtained by ultrathin sectioning in an ultramicrotome. These ribbons were collected in formvar-coated copper slot grids. After that, colloidal gold particles (10 nm) were deposited on both surfaces of the sections, being used as fiducial markers for the alignment of the tilted views. Single-axis tilt series (± 60° with 1° increment) were produced from samples using Xplore3D software and a Tecnai-G2 (FEI Company, Eindhoven, Netherlands) electron microscope operating at 200 kV. 3D reconstruction was performed using the IMOD software package 27 . Furthermore, tomogram generation by R-weighted back-projection was performed using ETOMO, and virtual slices were manually segmented using 3DMOD, which was also used to produce 3D models.
Extraction, separation of neutral lipids, and free sterol analysis. Total lipids were extracted from control and drug-treated Leishmania amazonensis promastigotes to analyze the effects of SBC 39 and SBC 40 on the free sterol composition of the promastigotes, as described previously 17,28 . Neutral lipids were analyzed by MS, and mass spectra were obtained by electron ionization (EI) at 70 eV according to the protocol published previously 17,28 . The assignment of structures was based on relative chromatographic behaviors, as well as the characteristic fragmentation patterns in MS, and by comparison of the mass spectra with those available in the National Institute of Standards and Technology (NIST) Research Library located at the NIST Mass Spectrometry Data Center.
Calibration for cholesterol and ergosterol determination. Five calibration standards were prepared from the pure standard of cholesterol, ergosterol, β-sitosterol, stigmasterol, and 5α-ergosta-8(14)-en-3β-ol purchased from Sigma-Aldrich Co. Different calibration solutions were prepared using ethyl acetate as solvent. To quantify cholesterol and ergosterol, standards were used at different concentrations of 0.08, 0.10, 0.25, 0.50, and 1.0 mM to plot the standard curve. From each calibration solution, 1 μl was injected (run in triplicate) into the GC-MS system to achieve the regression plot of various concentrations versus their peak area. To estimate the level of endogenous sterol/cell in control and drug-treated cultures, the total areas of the corresponding chromatographic peaks were divided by the cell densities of the cultures.

Statistical analysis.
All the graphics in the figures were created using the means of three independent experiments, and the bars represent the standard deviations of the means. The statistical significance of differences among the groups was assessed using the one-way or two-way analysis of variance (ANOVA) test, followed by Bonferroni's multiple-comparison test in the GraphPad Prism 5 software. Results were considered statistically significant when P was < 0.05(*), < 0.01(**), and < 0.001(***).

Results
The cytotoxic and antiproliferative effects of benzyl farnesyl amine mimetics SBC 37-43 against macrophages and Leishmania amazonensis. Figure 2 shows the cytotoxic effects of seven benzyl farnesyl amine mimetics SBC 37-43 by MTS/PMS assay of Leishmania amazonensis promastigotes. Six of them had CC 50 lower than 5 µM (SBC 37, SBC 39, SBC 40, SBC 41, SBC 42, and SBC 43), indicating an excellent effect against Leishmania amazonensis. After that, these six inhibitors were analyzed against Leishmania amazonensis promastigotes to evaluate their potential antiproliferative effects. Figure 3 shows the growth curves of the most potent inhibitors ( We also evaluated the cytotoxic effects of the SBCs 37-43 against murine macrophages using MTS/PMS assay after 72 h of treatment. Figure 4 shows that SBC 37, 38, 39, and 40 presented low cytotoxicity to mammalian cells, with CC 50 values of 33.94 µM, 40.53 µM, 40.65 µM, and 39.15 µM, respectively. Based on these results obtained for macrophages and promastigotes, we decided to evaluate the effects of three of them (SBCs 37, 39, and 40) against intracellular amastigotes. Figure Figure 6A shows a control L. amazonensis promastigote without any alteration in the morphology of the cell body, surface, and flagellum (Fig. 6A). Treatment with lower concentrations of SBCs induced several alterations, such as the presence of parasites rounded and swollen ( Fig. 6B,C,F), also presenting vesicles budding from the region near the flagellar pocket (Fig. 6E) and sometimes more than two flagella (Fig. 6C,D). After 48 h of treatment with 300 nM SBC 40, all parasites were completely rounded (Fig. 6F). These results indicate the potent effect of these inhibitors on altering the morphology of promastigotes. Furthermore, both inhibitors could induce lipid bodies accumulation after 48 h of treatment (Fig. 8). However, for SBC 39, the concentration to increase the accumulation of lipid bodies was 3-times higher than those for SBC 40. For the treatment with SBC 40, in lower concentrations, the ultrastructural alterations were similar to those obtained with SBC 39. In addition, a significant accumulation of lipid bodies randomly distributed throughout the cytosol was observed (Fig. 10C,D). The presence of glycosomes was easily observed in ultrathin sections of treated promastigotes, probably indicating an increasing number of them since they are difficult to observe in control parasites (Fig. 10C). Furthermore, several extracellular vesicles inside the flagellar pocket (Fig. 10D) and the presence of autophagosome-like vacuoles in close association with the nucleus and mitochondrion (Fig. 10A,E,F) were observed. In addition, alterations in the trans-Golgi network (Fig. 10E), disorganization of the kinetoplast (Fig. 10B), and intense mitochondrial swelling (Fig. 10B,F) were also induced by the treatments.
To better understand the ultrastructural effects induced by SBs, we decided to carry out treatments with high concentrations and a short incubation time. For this, we used a concentration of 5 µM for just 6 h. The results obtained indicated significant alterations showing that these new compounds presented a potent activity against Leishmania amazonensis. After treatment, plasma membrane projections were observed with 5 µM SBC 39 ( Fig. 11B-D, arrowhead). Interestingly, these projections appeared frequently in regions of the membrane close to the endoplasmic reticulum (Fig. 12D, arrowhead). Therefore, we decided to observe these projections by electron tomography. Figure 12A-D  www.nature.com/scientificreports/ treated with 5 µM SBC 39 for 6 h. From these sections, a specific area of the parasite surface was reconstructed (Fig. 12E,F), revealing the ultrastructure of this plasma membrane projection. Images confirmed the presence of the endoplasmic reticulum profile near the projection and the absence of microtubules associated with it. The alignment of the electron tomograms allows us to observe the relationship between the projection and the endoplasmic reticulum (Suppl. Figures 1 and 2-movies). Furthermore, the treatment with SBC 39 and SBC 40 for a short time caused several other alterations in the ultrastructure of the promastigotes, such as: mitochondrial swelling with an increased number of mitochondrial cristae (Fig. 11B,C,F); the presence of large vacuoles and myelin-like figures (Fig. 11B-F); and disorganization of the kinetoplast (Fig. 12E). After the alignment of several electron tomograms, it is possible to observe some of these changes in a movie containing a large volume of one promastigote treated with 5 µM SBC 39 (Suppl. Figure 3-movie).

Discussion
Although leishmaniasis has several treatment options, its therapy has a lot of problems such as extensive toxicity, lack of efficacy, parenteral route of administration affecting compliance, high costs, and emerging drug resistance 29 . Moreover, visceral, cutaneous, and mucocutaneous leishmaniasis remains some of the most devastating neglected tropical diseases. Thus, there is an urgent need to develop new anti-leishmanial compounds with more efficacy, low toxicity, and cost, which are preferentially administrated by oral or topic routes.
Sterol biosynthesis (SB) is an essential metabolic pathway in Leishmania sp. For many years, several SB inhibitors have been studied against both developmental stages of the parasite in vitro [15][16][17]23,28,[30][31][32] . Some of them have also been tested in vivo against Trypanosoma cruzi, inducing a potent suppressive effect in acute Chagas' disease 30,[33][34][35] . An important step of the SB is catalyzed by the enzyme squalene synthase (SQS). SQS is responsible www.nature.com/scientificreports/ for the reaction that catalyzes the first committed step in the SB pathway, thus, not interfering with isoprenoid production and its metabolites 11,36 . Several squalene synthase (SQS) inhibitors, such as quinuclidine derivatives, have been studied as potent SB inhibitors. The first quinuclidine derivatives tested against Leishmania sp. were BPQ-OH, ER-119884, and E5700, inducing cell death associated with the depletion of the parasite's endogenous sterols 23,31 . Furthermore, another quinuclidine derivative, WSP 1267, showed a potent effect against Candida albicans, C. parapsilosis, and C. tropicalis, with a MIC 50 of 2 µg/ml 37 . Some quinuclidine derivatives were also able to inhibit the recombinant L. major SQS at submicromolar concentrations, exhibiting selectivity action for the parasite enzyme 20,21 . Benzyl farnesyl amine mimics were also reported to be selective inhibitors of human  www.nature.com/scientificreports/ SQS. They have been explored for their potential use in developing cholesterol-lowering treatment options for hypercholesterolemia in men 38 .
In this study, we report the activity of several benzyl farnesyl amine mimetics against Leishmania amazonensis. The compounds SBC 39 and SBC 40 showed the most pronounced effects on the growth of L. amazonensis intracellular amastigotes, associated with low cytotoxicity in mammal cells and a higher selectivity index of 117.7 and 180, respectively. These selectivity indexes are higher than those found after treatment with posaconazole and itraconazole, two potent inhibitors of the growth of L. amazonensis 16 . Herein, the antiproliferative activities of SBC 39 and SBC 40 were in the nanomolar range against both extracellular promastigotes and intracellular amastigotes. Furthermore, the biological activity against Leishmania amazonensis was lower than those found for E5700 and ER-119884, two SQS inhibitors from Eisai Pharmaceutical Company, previously studied against Leishmania 17,26 . Nevertheless, it is noteworthy to mention that further development of E5700 and ER 119884 has been stopped because it caused testicle atrophy in a small animal experiment (personal communication). Beyond this, quinuclidine derivatives often bear a certain risk of neurological side effects (neurotoxicity of antimalarial drug quinine and other quinuclidine-containing drugs). Therefore, medicinal chemistry is increasingly interested in avoiding the quinuclidine moiety in early phase drug development. Finally, benzyl farnesyl amine mimetics have a much lower production cost, which is especially highly attractive for anti-infectious drug design in tropical emerging countries, where cost restrictions sometimes limit pharmaceutical development. Thus, these www.nature.com/scientificreports/ results should be regarded as a precious contribution to SB inhibitors research in tropical parasites with a high potential for further drug development. We used scanning and transmission electron microscopy to analyze morphological and ultrastructural alterations. Some morphological alterations such as the swelling of the cell body and the appearance of cells with several flagella were observed. Although the number of flagella in treated promastigotes was altered, arrest of the cell cycle was not observed, which is also found for other SB inhibitors 16,17,28 . Transmission electron microscopy images indicated that mitochondrion ultrastructure was dramatically altered after treatment with SBC 39 and SBC 40 (Figs. 9, 10). Fluorescence markers for mitochondrial membrane potential and ROS production confirmed the mitochondrial damage provoked by the treatments (Fig. 7). Alterations in the mitochondrion structure and function could be related to significant changes in the lipidic composition of the mitochondrial membranes since previous studies showed that the unique and ramified mitochondrion of the trypanosomatid has a particular composition of 24-methyl sterols 14 . Interestingly, in some images, several glycosomes were observed after treatment with SBC 40 (Fig. 10C), which could indicate an effort of the treated parasite to supply the mitochondrial damages and a possible decrease in the oxidative phosphorylation. An increasing number of glycosomes could help the parasite compensate for ATP production using the glycolytic pathway. www.nature.com/scientificreports/ Another important alteration was observed in the plasma membrane (Fig. 6), which could result from 24-methyl sterols depletion replaced by toxic intermediates of the sterol biosynthesis. This phenotype was also observed after treatment with several other SB inhibitors in Leishmania [15][16][17]26,27,38 . Moreover, several projections on the plasma membrane were observed after only 6 h of treatment, which was better observed by electron tomography (Figs. 11, 12). Projections could also be related to the secretory pathway since in some images, as shown in Fig. 10D, we observed several extracellular vesicles secreted by treated promastigotes. In addition, the presence of several giant vacuoles containing portions of the cytosol, damaged organelles, and membranes that could be related to an intense autophagic process, also indicating an increase in the secretory activity by the treated promastigotes.
Furthermore, the results (Tables 1, 2) indicated that both compounds (SBC 39 and 40) caused a concentrationdependent reduction in the ratio of endogenous 24-alkyl sterols (fecosterol, episterol, and 5-dehydroepisterol) to cholesterol (exogenous) below a critical level as in previous studies involving other SQS inhibitors 17,23 . For    www.nature.com/scientificreports/ instance, the ratio of cholesterol content to endogenous sterols in promastigotes control was 1:43. In contrast, in promastigotes treated with SBC 40 (at 300 nM), the cholesterol to endogenous sterols ratio changed dramatically to 1:7. The result is consistent with a blockade of de novo sterol synthesis at the level of SQS. Thus, the results suggest that both SBC 39 and SBC 40 have potent antiproliferative and selective growth inhibition effects on Leishmania amazonensis, and remarkably, they both have a dual mode of action (Fig. 14). The first is its interference in de novo sterol biosynthesis at the SQS level (Pre-Squalene Pathway); the second is the inhibition of downstream sterol biosynthesis, specifically in the isomerization of the ∆8 → ∆7 ***double bond and in the formation of the double bond between C-5 and C-6 of the ring B of the sterol (Post-Squalene Pathway).
In conclusion, our results support the notion that SBC 39 and SBC 40 are promising new chemotherapeutic agents against Leishmania sp. since they presented a very high specificity for the parasite. Furthermore, our findings justify future studies to better understand the mode of action and use in combination therapy with other SB inhibitors as a new therapeutic strategy that could reduce toxicity but increase the efficacy of treatment.  Table 2. Cholesterol and endogenous sterols a contents in control and SBC-treated L. amazonensis promastigotes. Mean and standard deviations (SD) were calculated from three parallel measurements (n = 3). a Endogenous sterols = Ergosta-8,24(24′)-trien-3β-ol, 5-dehydroepisterol and episterol. b The cholesterol:endogenous sterols ratio.